Structures and molecular mechanisms of RAD54B in modulating homologous recombination

Area of Science:

• Molecular Biology
• Genetics
• Structural Biology

Background:

• Genome stability is vital for cell survival, constantly challenged by DNA damage.
• DNA double-strand breaks (DSBs) are particularly dangerous and repaired via homologous recombination (HR) in S/G2 phases.
• RAD51 recombinase forms filaments for HR, regulated by accessory factors like RAD54B, whose functions are poorly understood.

Purpose of the Study:

• To elucidate the molecular mechanisms and functions of RAD54B in homologous recombination (HR).
• To define RAD54B's role in regulating RAD51 filament dynamics and DNA repair processes.

Main Methods:

• Cryo-electron microscopy (cryo-EM) to determine structural complexes.
• Site-directed mutagenesis to identify key functional domains.
• Biochemical assays to assess protein activity.
• Cellular assays to evaluate DNA repair in human cells.

Main Results:

• RAD54B stabilizes RAD51-DNA filaments, inhibits RAD51 ATPase activity, and promotes strand invasion and D-loop formation.
• The N-terminal domain (NTD) supports filament stabilization and strand exchange, while the ATPase domain is crucial for D-loop formation.
• A unique β-domain in RAD54B bridges RAD51 protomers, contacts dsDNA, and regulates RAD54B's ATPase activity and oligomerization.
• RAD54B NTD and β-domain are essential for repairing camptothecin-induced DSBs via HR in human cells.

Conclusions:

• RAD54B possesses a modular architecture critical for its function in HR.
• The study reveals a mechanistic framework for RAD54B's role in stabilizing RAD51 filaments and facilitating DNA repair.
• RAD54B is essential for genome maintenance by ensuring efficient homologous recombination repair of DSBs.